Skin is a complex, multi-layered and dynamic system that is the largest organ of the body and is vitally important to both our health and self image. Skin, which comprises three principal layers (e.g., the epidermis, the dermis, and the hypodermis or subcutis layer), contains a wide variety of cellular types and structures, including epidermal and dermal connective tissue with blood and lymphatic vessels, hypodermal adipose tissue, and the elastic fascia beneath the hypodermis. In turn, these structures are composed of a number of different cellular types including keratinocytes, melanocytes, fibroblasts, endothelial cells, and adipocytes.
Skin aging is a complex, multi-factorial process that can result from unrepaired cellular and tissue damage, which can be caused by a variety of intrinsic and extrinsic factors occurring over decades. Some of the biological themes/pathways believed to be involved with aging skin include lipid biosynthesis, epidermal cell differentiation, extracellular matrix organization and biogenesis, wound healing, immune response, and inflammatory response. See, e.g., Genomics of Skin Aging: Practical Applications, Journal of Drugs in Dermatology Supplement, Vol. 8, Issue 7 (2009). Over time, skin aging can result in, for example, the appearance of fine lines and wrinkles (an example of which is crow's feet in the periorbital area), damage to skin barrier properties resulting in skin dryness, skin sagging, a reduction in skin strength and elasticity, and so forth.
Given the significant impact that skin aging can have on one's appearance and self esteem, there is an on-going desire to identify cosmetic agents that are effective at treating or improving the appearance of aging skin. Skin models capable of mimicking aspects of cellular processes integral to skin aging are therefore desired in order, for example, to identify these agents. Modeling techniques to study skin physiology and skin responses to agents have historically included a variety of specific techniques, from the culturing of a single cell type or a small number of co-mingled cell types, to fabricating human tissue equivalents, to developing animal models. However these relatively simple models often lack much of the intra and intercellular complexities of human skin. For example, cell cultures of single cell types are easily utilized but overly simplistic and can have limitations for generalizing results to human skin First, such cultures contain cells that are altered simply by being cultured and, in some cases, have been altered by genetic manipulation to promote easy cell passaging and maintenance. Second, such cultures may not account for intrinsic intricate matrices of cells constantly interacting as a unit. Multi-celled cultures can be limited due to difficulties in creating the integrated mechanical structure of native tissue and in ensuring that the cells comprise the extracellular components necessary to maintain cellular genetic expression levels at a normal physiological level. Models that include stem cells treated to mimic human skin are suitable for their intended purposes, but can also fall victim to similar concerns and limitations.
More complex skin models include animal models and skin-equivalent models. While having additional complexity, animal models can suffer from limitations including the genetic variation with respect to human skin; in the analysis of obtained results there can be a concern that human tissues react differently from animal tissues and the ability to generalize results may be compromised.
Skin-equivalent models can be limited by lack of cellular interconnectivity, permeability concerns, and anatomical simplicity. Some recent attempts at skin-equivalent models may be described as organotypic human tissue equivalents and include in vitro reconstructions of human cells such as keratinocytes cultured on an inert polycarbonate filter. These models by their very nature may be limited in that they can have reduced barrier function that can lead to aberrant sensitivities to tested agents. The models may also be less complex than human skin, having perhaps one or two cells types (such as keratinocytes and fibroblasts or keratinocytes and melanocytes) but lacking additional cells such as endothelial cells or even the full keratinocyte, fibroblast, and melanocyte combination. In addition the organotypic skin equivalent models may also be missing normal skin structures such as glands that may affect skin response.
The most complex skin model involves the ex vivo culture of human skin tissue samples. Some previous attempts at such models included small biopsies of skin floating directly in media. It is known in the art that transient cultures may be deficient, as inventors and researchers have indicated attempts at ex-vivo pig skin grafts are limited to seven days (Vielhaber et al., Ex vivo Human Skin Model, US2009/0298113). Attempts at improving the longevity of ex-vivo skin have been sought, one example being described in EP 2 019 316 B1.
Even more recent ex-vivo models have involved attempting to culture skin explants on metal grids (Mitts et al., Elastin Protective Polyphenolics and Methods of Using the Same, US2009/0110709) and skin grafting to the chorioallantoic membrane (CAM) of a fertilized ovarian egg (Goldstein et al., Chimeric Avian-Based Screening System Containing Mammalian Grafts, US2009/0064349). However such models may be still limited by transiency of the construct, delicacy, and even xenogeneic concerns.
Despite these advancements, a need continues to exist for a sensitive and predictive ex-vivo human skin tissue screening methods and models suitable for screening tens or hundreds of compounds for select activity in a large number of tissue donors. However, there are many challenges/uncertainties associated with development of ex-vivo tissue culture models and methods that are predictable and repeatable for large numbers of test compounds across a large tissue donor population. Some non-limiting examples include one or more of: the effect of donor to donor variability, whether ex-vivo skin tissue can be properly regulated over the requisite culturing time periods, whether analysis of gene transcriptomics, proteomics, and/or metabomics of ex-vivo skin tissue can be predictive of in vivo results, whether appropriate positive controls could be identified for use in a high enough percentage of ex-vivo tissue samples to develop a satisfactory screening method, whether culturing conditions could be identified that enabled a sufficiently robust tissue response in a high enough percentage of ex-vivo tissue samples, and whether these variables could be controlled to the point that the effects of a test agent on an ex-vivo skin tissue sample could be isolated and interpreted.